Lecture 22

Phase Changes

As the sporophyte develops it passes through different phases. We have already discussed the embryonic phase which ended in seed maturation and developmental arrest. Upon germination, the plant enters the juvenile phase, followed by an adult phase and flowering. The most obvious and dramatic post-germinative phase change is flowering but vegetative phases can be quite distinct in some plants.

 

Vegetative Phases

Vegetative development is divided into juvenile and adult phases. Juvenile plants are not competent to flower while adult phase plants have gained the competence to flower. This has economic ramifications in many crops, including fruit trees where trees cannot bear fruit until reaching the adult phase. There are also morphological and physiological differences between juvenile and adult which are not obvious in all species. Some of these represent plant adaptations for survival. For example, some woody shrubs protect their succulent juvenile tissues from grazing by accumulating bitter secondary compounds.

Juvenile Phase represents the early phase of vegetative development, therefore the juvenile organs are the first organs produced. They will be at the bottom of the plant and will be the oldest organs of the plant. Adult phase represents a later stage of development so adult organs will be at the top of the plant and younger in age.

Examples of vegetative phase differences

Ivy—The juvenile grows as a prostrate vine with alternate phyllotaxy while the adult grows as an upright shrub with spiral phyllotaxy.

Eucalyptus—the juvenile has opposite phyllotaxy with short broad leaves, the adult has spiral phyllotaxy with long slender leaves.

Maize—juvenile has short round leaves covered with epicuticular wax and no epidermal hairs, adult has long narrow leaves with no epicuticular wax but with epidermal hairs.

Regulation of vegetative phases

The hormone gibberellic acid (GA) promotes juvenility in some plants. For example in ivy, if the apical bud is removed from an adult branch, the subtending axillary buds will grow into typical adult branches. However, if GA is applied to the axillary buds they will grow into a juvenile branch. In maize, GA deficient mutants are delayed in their transition to adult phase.

Dominant gain-of-function maize mutations, called Teopod, prolong the juvenile phase but do not affect the onset of adult characteristics. In other words, there are leaves that normally would display adult characteristics which now display juvenile and adult simultaneously. This indicates that the Teopod genes regulate phase change by promoting juvenility and also that juvenile and adult phases are regulated independently. Genetic mosaic experiments where sectors of wild type tissue were induced in Teopod mutant plants show that the Teopod gene acts through some diffusible substance. In leaves or stems that were half mutant and half wild type, the wild type tissue showed the mutant characteristics.

 

Floral Induction

Upon reaching adult phase, plants are competent to flower (enter reproductive phase). The process of floral determination is called floral evocation. Some plants flower because of intrinsic signals while others require environmental cues. The most common environmental cues are temperature and photoperiod.

Vernalization

Many biennial plants and "winter crops" such as winter wheat require a cold period to flower. An exposure to cold that stimulates flowering is called vernalization. Different plants have requirements for different vernalization temperatures and periods. In most plants vernalization can be reversed by warm temperatures and  so the cold period must be relatively uninterrupted.

The site of cold perception is the SAM. A SAM grafted from an unvernalized plant onto one that has received cold treatment will not flower while a SAM grafted from a vernalized plant onto an unvernalized one will flower.

Photoperiod

Plants are classified as short day, long day or day neutral according to their photoperiod requirements. Short day plants flower under regimes of short day, long night while day neutral plants have no specific photoperiod requirements. In addition plants can be classified as obligate or facultative in their photoperiod requirements. Obligate plants will remain vegetative indefinitely without the inductive photoperiod while facultative plants will eventually flower without the inductive photoperiod but can be induced to flower sooner. (Plants can be obligate or facultative with regard to vernalization requirements also).

Long day plants have a minimum required light period for photoinduction; anything above that will result in flowering and there is no dark period requirement. Short day plants have a maximum photoperiod over which they will not flower; they require a dark period. These are more accurately described as long night plants because it is the dark period that is inductive. The darkness must be continuous. Interrupting the dark period with a flash of light prevents photoinduction.

Different plants have different requirements for the number of photoinductive cycles. Some require only one cycle, others require several weeks. Once a plant is induced, placing it in noninductive cycles will usually not reverse the photoinduction.
Photoperiod is perceived through the photoreceptor phytochrome. Phytochrome is a protein with a covalently attached chromophore. Phytochrome exists in 2 interconvertable forms. Pr absorbs light in the red part of the spectrum (~660 nm). Pfr absorbs far red light (~730 nm). Absorption of red light converts Pr to Pfr and absorption of far red light converts Pfr to Pr. Pfr also undergoes spontaneous "dark reversion" to Pr. Daylight has a relatively high proportion of red light and thus most phytochrome is in the Pfr form. In the dark, the equilibrium slowly shifts to Pr. This cycling of phytochrome somehow interacts with the circadian clock to determine the proper seasonal time for flowering.

The site of photoperiod perception is the leaves. Exposure of the SAM to photoinductive cycles has no effect. In cocklebur (a short day plant), exposure of 8 cm2 of leaf surface to short days while the rest of the leaves receive long days induces flowering. Not all leaves are equally receptive. Juvenile leaves cannot perceive or transmit the photoinductive signal. The first adult leaves may require more photoinductive cycles to induce flowering than later adult leaves. Grafting experiments show this is a property of the state of the leaf, not it’s position relative to the SAM. Early adult and late adult leaves grafted onto identical positions of recipient plants will still require different numbers of photoinductive cycles.
The induced state is stable in the leaf. A Perilla leaf that has been photoinduced can be grafted to an uninduced plant and cause flowering. The same leaf can then be removed and regrafted several times to other plants under noninductive conditions and continue to induce flowering.

SAMs from different phases differ in their competence to respond to floral induction. Grafting juvenile meristems of tobacco onto florally induced plants did not produce flowering until after a period of growth (presumably until after adult phase transition) while grafting adult meristems onto induced plants resulted in rapid flowering. The veg gene in pea is required to confer floral competence to SAMs. veg mutant SAMs grafted onto wild type plants under inductive photoperiods will not flower, however wild type SAMs grafted onto veg mutant plants will.

Florigen: Because of the hormonal nature of the signal sent from the leaves to the SAM, it was believed that a floral inducing hormone existed. This was called florigen. It has never been isolated and current opinion favors a more complex explanation than just a single substance.

Increased hormone sensitivity is correlated with floral induction

Several lines of evidence, including culturing meristems with various levels GA or GA inhibitors suggest that an increased sensitivity of the SAM to GA is associated with floral induction. Recently, a gene called FPF1 (floral promoting factor) was identified in Arabidopsis. FPF1 mRNA is not present in the vegetative SAM but is induced in the SAM at the onset of floral induction. Constitutive expression of FPF1 leads to an early flowering phenotype. In addition, plants show additional phenotypic aspects like elongated internodes that are reminiscent of GA responses. The FPF1 overexpressors do not show early flowering or internode elongation if combined with GA deficient mutants or treatment with paclobutrazol (a GA inhibitor). Therefore these effects of FPF1 are GA dependent indicating that expression of FPF1 increases sensitivity to GA and that this increased sensitivity of the SAM leads to floral induction. Consistent with the role for GA in floral induction is a report that the LEAFY (a gene involved in arabidopsis flower development) promoter is GA responsive.

 

Floral Determination

Day-neutral tobacco consistently forms about 40 nodes and then a terminal flower. Undetermined vegetative SAMs can be cut off and rerooted indefinitely. Removal of leaves from plants has no effect on the number of nodes to flowering, but induction of adventitious roots by piling up soil as the stem grows increases the number of nodes produced. This suggests that the distance between the SAM and roots is important for floral determination. SAMs become florally determined prior to actual floral development. Determined SAMs that are cut off and rerooted will produce 4 more nodes, then a flower (the same as if they had remained on the plant).

Subtending axillary buds also form floral branches in tobacco. The axillary buds form over a period of 5 days following SAM floral determination. The earliest the axillary buds are large enough to remove and root is 9 days after. At this time most but not all axillary buds are florally determined. Thus floral determination in lower axillary buds occurs later than in the terminal meristem.

The florally determined state is not restricted to tissues that will actually form flowers. Internode segments will produce shoots when cultured on hormone free medium. Vegetative state segments will produce vegetative shoots while segments from the upper nodes of flowering plants will form floral shoots.

Sunflower and maize represent extremes in the timing of floral determination. Rooting experiments showed that the sunflower SAM is determined to flower during the seedling stage, approximately 14 nodes prior to morphological differentiation. The apical bud was removed and rooted at different times. Whether the apex was removed from the seedling or after producing 9 nodes, the total number of nodes produced before flowering remained about 16.

In maize, floral determination does not occur until after vegetative development is complete and it appears to be a stepwise process. Cultured SAMs form vegetative shoots until floral transition. Early tassel meristems develop vegetative shoots from their branch primordia. Later tassel primordia develop sterile flowers with leaf like organs. Only flowers that are initiating floral organs at the time of culture will go on to complete normal floral development.

 

Genetic Control

Arabidopsis is a facultative long day plant with a facultative response to vernalization. Genes that regulate flowering have been identified in genetic screens for mutants that are either early flowering or late flowering relative to wild types under the same set of conditions. Early flowering mutants identify genes that inhibit flowering, late flowering mutants define genes that promote flowering (or inhibit inhibitors). At least 2 independent pathways function. The photoperiod dependent pathway is defined by mutants that flower late only under inductive photoperiod but flower at the same time as wild type under non-inductive photoperiod. Mutants with altered flowering times under non-inductive conditions are involved in the “autonomous” pathway.

Genes of the autonomous pathway include LUMINODEPENDENS (LD), FRIGIDA (FRI)  and FLOWERING LOCUS C (FLC). FLC encodes a Mads box transcription factor. FLC expression is positively regulated by FRI and negatively regulated by LD and by vernalization.

Genes of the photoperiod sensitive pathway include CONSTANS which encodes a zinc finger transcription factor.

 

Measuring developmental time

Because of the nature of plant development, spatial relationships change over time. For example the SAM becomes increasingly separated from the root system over time. This makes it difficult to tell whether the mechanisms regulating the duration of developmental phases are based on temporal or spatial considerations. Thus there is debate over whether plant homeotic mutants are really heterochronic. Homeotic is when structures form in an inappropriate place. Heterochronic is when something occurs at an inappropriate time. This dilemma is particularly evident in the control of phase changes. Phase change could be based on spatial relationships (distance between the SAM and the root system for example) or on temporal considerations or counting mechanisms. The phenotype of one mutant in arabidopsis called paused has been interpreted to suggest there might be a "developmental clock." paused plants initiate leaves more slowly than normal. The first seedling leaf after germination is delayed by several days relative to wild type. The first leaf that forms in the mutant resembles the leaf that the normal plant would be making at the same time although it is not in the same position. Furthermore, this mutant flowers at the same time as normal even though it possesses fewer total leaves. Thus it appears to enter phase transitions at the proper time, regardless of position. There are other possible explanations and the majority of the evidence (from rooting experiments etc.) suggests that spatial relationships are more important for determining phase.